CN117761304A - Dynamic dual-mode and ultrasensitive immunodetection test strip and preparation method thereof - Google Patents
Dynamic dual-mode and ultrasensitive immunodetection test strip and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of immunodetection, and particularly discloses a dynamic dual-mode ultrasensitive immunodetection test strip and a preparation method thereof. The method comprises the following steps: after carrying out surface chemical modification on MAF nano particles, mixing and incubating with AuNPs, self-assembling to form MAF@AuNPs composite nano particles, further mixing and incubating with Ab1, adding a sealing agent for continuous incubation, centrifuging, taking precipitates, re-dissolving by using a re-solvent, preparing Ab1 modified MAF@AuNPs composite nano particles, dripping the Ab1 modified MAF@AuNPs composite nano particles on a marking pad, and drying; streaking Anti-Ab1 on a chromatographic membrane, and marking as a C line; drawing Ab2 on a chromatographic membrane, marking as a T line, and drying; the sample pad was treated with borate buffer to prepare an immunodetection test strip. The invention realizes the dynamic complementation of fluorescence-colorimetric dual-mode signals, and the fluorescence mode sensitivity is improved by more than 100 times compared with the color mode sensitivity.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to a dynamic dual-mode ultrasensitive immunodetection test strip and a preparation method thereof.
Background
The immune detection test strip is an in-vitro diagnosis technology with the most extensive application, has the advantages of rapidness, convenience, low cost and naked eye visualization, and the practical application scene of the immune detection test strip is gradually expanded from the high-concentration target detection requirement (ng/mL-ug/mL) to the low-concentration target detection requirement (pg/mL-ng/mL). Most of the existing immunodetection test strips are based on AuNPs or fluorescent microspheres/quantum dots, adopt single-mode colorimetric or fluorescent signals for detection, and face the key problems that: the AuNPs immune detection test strip is easy to identify by naked eyes and quantitatively analyze when detecting a high-concentration target, but the sensitivity is insufficient, so that the low-concentration target is difficult to effectively detect; the sensitivity of the fluorescent microsphere/quantum dot immune detection test strip is greatly improved, and the low-concentration target can be effectively detected, but the fluorescent signal is easy to saturate when the high-concentration target is detected, and the fluorescent microsphere/quantum dot immune detection test strip is difficult to effectively identify by naked eyes or quantitatively analyze.
At present, a small amount of researches report that a fluorescence-colorimetric dual-mode immunodetection test strip based on a fluorescence quantum dot@AuNPs composite material realizes fluorescence-colorimetric dual-mode detection signal reading, but the sensitivity of a fluorescence signal mode is not remarkably improved compared with that of a color signal mode and is still in the same order of magnitude, on one hand, dynamic naked eye identification and simultaneous ultrasensitive quantitative analysis covering high and low concentrations cannot be realized, on the other hand, the preparation of the research report fluorescence quantum dot@AuNPs composite material is relatively complex based on a layer-by-layer coating stepwise synthesis method, and the follow-up antibody protein labeling and the like still depend on the traditional chemical covalent modification technology, so that the technological difficulty and the cost of manpower and material resources are increased, and the low cost economy of the immunodetection test strip technology is reduced.
In order to meet the application requirements of different detection scenes and solve the problem of complex preparation process. Therefore, it is necessary to develop a dynamic dual-mode ultrasensitive immune detection test strip and a preparation method thereof, which can realize ultrasensitive detection in a fluorescent signal mode when a target is low in concentration and can realize effective naked eye identification and quantitative analysis in a colorimetric signal mode when the target is high in concentration.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a dynamic dual-mode ultrasensitive immunodetection test strip and a preparation method thereof, which can realize ultrasensitive detection in a fluorescent signal mode when a target is low in concentration and can realize effective naked eye identification and quantitative analysis in a colorimetric signal mode when the target is high in concentration.
The first aspect of the invention provides a method for preparing a dynamic dual-mode ultrasensitive immunodetection test strip.
Specifically, the method comprises the following steps:
(1) Sequentially fixing the bottom plate, a water absorption pad, a chromatographic membrane, a marking pad and a sample pad on the bottom plate;
(2) Using tetra (4-carboxylbenzene) ethylene as an organic ligand and zirconium chloride as metal ion salt to synthesize MAF nano particles;
(3) Coating the nano gold particles with sodium citrate to prepare AuNPs;
(4) After carrying out surface chemical modification on MAF nano particles, mixing and incubating the MAF nano particles with AuNPs for 0.5-2 h, and self-assembling to form MAF@AuNPs composite nano particles;
(5) Mixing and incubating MAF@AuNPs composite nano particles with primary antibody protein (Ab 1) for 1-3 hours, adding a sealing agent for further incubation for 0.1-1 hour, centrifuging, taking the precipitate, and re-dissolving by using a re-solvent to prepare Ab1 modified MAF@AuNPs composite nano particles;
(6) Dripping Ab1 modified MAF@AuNPs composite nano particles on a marking pad, and drying;
(7) Streaking an Anti-protein antibody (Anti-Ab 1) on a chromatographic membrane, and marking the chromatographic membrane as a C line; drawing a secondary antibody protein (Ab 2) on a chromatographic membrane, marking as a T line, and drying;
(8) The sample pad was treated with borate buffer to prepare an immunodetection test strip.
Preferably, in the step (2), the particle size of the MAF nanoparticle is 150 to 250nm.
Further preferably, the particle size of the MAF nanoparticle is 200nm.
Preferably, in the step (5), 1 to 3 parts by volume of MAF@AuNPs composite nano particles are provided, 0.02 to 0.05 part by volume of primary antibody protein is provided, 0.1 to 0.4 part by volume of blocking agent is provided, and 0.2 to 0.5 part by volume of complex solvent is provided.
Preferably, in the step (7), the primary antibody is 2-5 parts by weight, and the secondary antibody is 2-5 parts by weight.
Preferably, in the step (3), the particle size of the AuNPs is 10 to 30nm.
Further preferably, the particle size of the AuNPs is 15nm.
Preferably, in step (4), the step of chemically modifying the surface comprises: and mixing and incubating the MAF nano particles and positively charged polymer molecules for 0.1-1 h, centrifuging, and taking the precipitate for redissolution.
Further preferably, the step of chemically modifying the surface comprises: and mixing and incubating the negatively charged MAF nano particles and positively charged polymer molecules for 0.5h at room temperature under the condition of stirring, centrifuging the mixed solution, removing supernatant, taking precipitate, and redissolving the precipitate in the same volume of deionized water.
Preferably, in the step (4), after the surface chemical modification of the MAF nanoparticles, the MAF nanoparticles are mixed with AuNPs and incubated for 1h.
Further preferably, in the step (4), after carrying out surface chemical modification on MAF nano particles at room temperature under the condition of stirring, mixing and incubating the MAF nano particles with negatively charged AuNPs for 1h, centrifuging the mixed solution to remove supernatant, taking precipitate, and redissolving the precipitate in equal volume of deionized water to obtain the self-assembled MAF@AuNPs composite nano particles.
AuNPs are adsorbed on the surface of fluorescent MAF nano particles through electrostatic action, so that the traditional complex layer-by-layer wrapping synthesis process is avoided, the preparation process is effectively simplified, and simultaneously AuNPs are exposed to a solvent and can be used for directly adsorbing and marking antibody proteins, so that the traditional carboxyl/amination functional modification and covalent crosslinking are avoided, and the preparation process is further effectively simplified, and the economy is improved.
Preferably, the positively charged polymer molecule is at least one of polylysine, epsilon-polylysine, polyvinylamine, polyacrylamide, polyamide and polyethyleneimine.
Further preferably, the positively charged polymer molecule is a polyethyleneimine.
Preferably, the molecular weight of the polyethyleneimine is 600 to 20000.
Further preferably, the molecular weight of the polyethyleneimine is 600, 2000, 5000, 10000, 20000.
Still more preferably, the polyethyleneimine has a molecular weight of 10000.
Preferably, the mass concentration of the polyethyleneimine is 0.01-1%.
Further preferably, the mass concentration of the polyethyleneimine is 0.1%.
When the 0.1% polyethyleneimine-10000 is used for carrying out surface chemical modification on the MAF nano-particles and then is mixed with AuNPs for incubation, under the same condition, the input amount of the AuNPs determines the quantity of the AuNPs adsorbed on the surface of the single MAF nano-particles, wherein the input amount of the AuNPs is 1-5 parts by volume.
Preferably, the AuNPs is added in an amount of 2.5 parts by volume.
Preferably, in the step (5), the MAF@AuNPs composite nano particles and primary antibody protein (Ab 1) are mixed and incubated for 2 hours, then a blocking agent is added for further incubation for 0.5 hour, and after centrifugation, precipitation is taken and re-dissolved by using a complex solvent, so that the Ab1 modified MAF@AuNPs composite nano particles are prepared. When MAF@AuNPs composite nano-particles are mixed and incubated with Ab1, pH does not need to be adjusted.
Preferably, in step (5), the blocking agent and the cosolvent are phosphate buffers containing any one of bovine serum albumin, polyethylene glycol, gelatin and tween.
Further preferably, the blocking agent and the cosolvent are phosphate buffers containing bovine serum albumin.
Preferably, in the step (6), 0.001-0.003 parts by volume of Ab1 modified MAF@AuNPs composite nano particles are dropwise added to a marking pad, and the marking pad is dried.
Further preferably, in the step (6), 0.002.5 parts by volume of Ab1 modified MAF@AuNPs composite nano particles are dripped on the marking pad and dried.
A second aspect of the invention provides a dynamic dual mode, ultrasensitive immunodetection test strip.
Specifically, the immune detection test strip comprises a bottom plate, and a water absorption pad, a chromatographic membrane, a marking pad and a sample pad which are positioned on the bottom plate; the length of the immune detection test paper strip is 40-100 mm, and the width is 2-10 mm.
Preferably, the bottom plate is polyvinyl chloride.
Preferably, the water absorbing pad is filter paper.
Preferably, the chromatographic membrane is a nitrocellulose membrane; the chromatographic membranes are marked as C lines and T lines at intervals of 5-10 mm respectively.
Further preferably, the scribe marks are C lines and T lines with widths of 0.3-1.0 mm.
Still more preferably, the scribe marks are marked with C-lines and T-lines having a width of 0.35mm.
Preferably, the pore diameter of the chromatographic membrane is 6-10 μm.
Further preferably, the pore diameter of the chromatographic membrane is 8. Mu.m.
Preferably, the marking pad is glass fiber.
Preferably, the sample pad is glass fiber treated by the treatment liquid.
Further preferably, the treatment liquid is borate buffer.
The third aspect of the invention provides a method for detecting a dynamic dual-mode and ultrasensitive immune detection test strip.
Specifically, the detection method comprises the following steps:
and dripping the sample solution to be detected on a sample pad of the immunodetection test strip, dripping the working buffer on the sample pad, and observing a colorimetric signal of the T line/C line when the sample solution to be detected is in high concentration and observing a fluorescent signal of the T line/C line under ultraviolet light when the sample solution to be detected is in low concentration after the chromatography is finished.
Preferably, the chromatographic time is 10-15 min.
Further preferably, the chromatography is performed for 15min.
Preferably, the working buffer solution is deionized water solution containing Triton X-100, the addition amount of the working buffer solution is 0.01-0.05 part by volume, and the working buffer solution is used for supporting chromatographic flow.
Preferably, the wavelength of the ultraviolet light is 302nm.
Compared with the prior art, the invention has the following beneficial effects:
the immune detection test strip prepared by the invention realizes dynamic complementation of fluorescent-colorimetric dual-mode signals, can realize ultrasensitive detection by a fluorescent signal mode when a target is low in concentration, can realize effective naked eye identification and quantitative analysis by a colorimetric signal mode when the target is high in concentration, and has fluorescence mode sensitivity which is improved by more than 100 times compared with color mode sensitivity. The immune detection test strip has wide detection application range and covers conventional detection objects and ultrasensitive detection objects. The whole process of the preparation method is simplified, and the cost of manpower and material resources is saved; the AuNPs treated by the method can directly adsorb and mark antibody proteins, and avoid chemical modification and covalent crosslinking.
Drawings
FIG. 1 is a diagram of the results of a detection model of a PCT dynamic dual-mode and ultrasensitive immune test strip;
FIG. 2 is a graph showing the result of a D-dimer dynamic dual-mode and ultrasensitive immune test strip detection model;
FIG. 3 is a graph showing the result of a dynamic dual-mode, ultrasensitive immune test strip detection model of NT-proBNP;
FIG. 4 is a graph showing the result of a dynamic dual-mode and ultrasensitive immune test strip detection model of cTnI.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
The invention is characterized in that: in order to meet the application requirements of different detection scenes and solve the problem of complex preparation process. The invention discloses a fluorescent-colorimetric dual-mode dynamic and ultrasensitive immunodetection test strip, which is characterized in that fluorescent MAF nanoparticles and AuNPs are self-assembled through surface chemical design to form a MAF@AuNPs nanocomposite, the MAF@AuNPs nanocomposite has fluorescent and colorimetric dual-mode signals, auNPs in the self-assembled MAF@AuNPs nanocomposite are adsorbed on the surface of the MAF particles, so that complex synthesis process is avoided on one hand, and on the other hand, auNPs are exposed to the solution and can be used for directly adsorbing and marking antibody proteins subsequently, and the whole process is further simplified. The immune detection test strip based on MAF@AuNPs nano-composite realizes ultrahigh sensitivity of low-concentration target detection by using a fluorescence mode signal of MAF, realizes naked eye identification and efficient quantitative analysis of high-concentration target detection by using a colorimetric mode signal of AuNPs, wherein the sensitivity of the fluorescence mode signal is improved by more than 100 times compared with that of the color mode signal, and forms a dual-mode dynamic complementary instant detection system, and the requirements of various potential high-concentration target detection and low-concentration target detection are met.
Example 1
A dynamic dual-mode ultrasensitive immunodetection test strip and its preparation method are provided.
Example 1 is exemplified by Procalcitonin (PCT) assay.
The materials/reagents used in this example include, in parts by volume:
2 parts of MAF@AuNPs composite nano particles, 0.03 part of primary antibody protein PCT-Ab1, 0.2 part of blocking agent, 0.3 part of cosolvent and 0.04 part of working buffer.
The weight portion of the composition comprises:
2.5 parts of primary Anti-protein antibody Anti-PCT-Ab1 and 2.5 parts of secondary Anti-protein PCT-Ab2.
The preparation method comprises the following steps:
(1) Sequentially fixing the bottom plate, a water absorption pad, a chromatographic membrane, a marking pad and a sample pad on the bottom plate;
(2) Using tetra (4-carboxylbenzene) ethylene as an organic ligand and zirconium chloride as metal ion salt to synthesize MAF nano particles;
(3) Coating the nano gold particles with sodium citrate to prepare AuNPs;
(4) Mixing and incubating negatively charged MAF nano particles with 0.1% polyethylenimine-10000 for 0.5h at room temperature under stirring, centrifuging the mixed solution, removing supernatant, taking precipitate and redissolving in equal volume of deionized water to obtain surface chemically modified MAF nano particles, mixing and incubating the surface chemically modified MAF nano particles with 2.5 parts of negatively charged AuNPs for 1h, centrifuging the mixed solution to remove supernatant, taking precipitate and redissolving in equal volume of deionized water to obtain self-assembled MAF@AuNPs composite nano particles;
(5) Mixing MAF@AuNPs composite nano particles with PCT-Ab1, placing the mixture in a shaking table for incubation for 2 hours, adding a sealing agent for further incubation for 0.5 hour, centrifuging at 8000rpm for 10 minutes, removing supernatant, taking precipitate, and redissolving the precipitate by using a cosolvent to prepare PCT-Ab1 modified MAF@AuNPs composite nano particles;
(6) Dripping PCT-Ab1 modified MAF@AuNPs composite nano particles with the volume part of 0.0025 part into a marking pad, and drying;
(7) Streaking Anti-PCT-Ab1 on a chromatographic membrane, and marking as a C line; marking PCT-Ab2 on a chromatographic membrane, marking as T line, and oven drying;
(8) The sample pad was treated with borate buffer to prepare an immunodetection test strip.
The detection method comprises the following steps:
and (3) dripping a standard solution containing a target to be detected on a sample pad, dripping a corresponding working buffer on the sample pad, and respectively recording colorimetric and fluorescent signals of a T line/C line after chromatography is finished (15 min).
As shown in FIG. 1, the detection result shows that the Color mode (Color) sensitivity is 5ng/mL, the Fluorescence mode (Fluorescence) sensitivity is 10pg/mL (clinical threshold 50 pg/mL), and the Fluorescence mode is improved by about 500 times compared with the Color mode sensitivity.
Example 2
A dynamic dual-mode ultrasensitive immunodetection test strip and its preparation method are provided.
Example 2 is exemplified by the detection of D-dimer (D-dimer).
The materials/reagents used in this example include, in parts by volume:
2 parts of MAF@AuNPs composite nano particles, 0.03 part of primary antibody protein D-dimer-Ab1, 0.2 part of blocking agent, 0.3 part of cosolvent and 0.05 part of working buffer.
The weight portion of the composition comprises:
2.5 parts of primary Anti-protein antibody Anti-PCT-Ab1 and 2.5 parts of secondary Anti-protein PCT-Ab2.
The preparation method comprises the following steps:
(1) Sequentially fixing the bottom plate, a water absorption pad, a chromatographic membrane, a marking pad and a sample pad on the bottom plate;
(2) Using tetra (4-carboxylbenzene) ethylene as an organic ligand and zirconium chloride as metal ion salt to synthesize MAF nano particles;
(3) Coating the nano gold particles with sodium citrate to prepare AuNPs;
(4) Mixing and incubating negatively charged MAF nano particles with 0.1% polyethylenimine-10000 for 0.5h at room temperature under stirring, centrifuging the mixed solution, removing supernatant, taking precipitate, redissolving in equal volume of deionized water to obtain surface chemically modified MAF nano particles, mixing and incubating with 2.5 parts of negatively charged AuNPs for 1h, centrifuging the mixed solution to remove supernatant, taking precipitate, redissolving in equal volume of deionized water, and self-assembling to form MAF@AuNPs composite nano particles;
(5) Mixing MAF@AuNPs composite nano particles with D-dimer-Ab1, placing the mixture in a shaking table for incubation for 2 hours, adding a sealing agent for further incubation for 0.5 hour, centrifuging at 8000rpm for 10 minutes, removing supernatant, taking precipitate, and redissolving the precipitate by using a cosolvent to prepare the D-dimer-Ab1 modified MAF@AuNPs composite nano particles;
(6) Dripping 0.0025 part by volume of D-dimer-Ab1 modified MAF@AuNPs composite nano particles into a marking pad, and drying;
(7) Streaking an Anti-D-dimer-Ab1 on a chromatographic membrane, and marking the chromatographic membrane as a C line; marking D-dimer-Ab2 on a chromatographic membrane, marking as T line, and drying;
(8) The sample pad was treated with borate buffer to prepare an immunodetection test strip.
The detection method comprises the following steps:
and (3) dripping a standard solution containing a target to be detected on a sample pad, dripping a corresponding working buffer on the sample pad, and respectively recording colorimetric and fluorescent signals of a T line/C line after chromatography is finished (15 min).
As shown in FIG. 2, the detection result shows that the Color mode (Color) sensitivity is 20ng/mL, the Fluorescence mode (Fluorescence) sensitivity is 100pg/mL (clinical threshold 500 ng/mL), and the Fluorescence mode is improved by about 200 times compared with the Color mode sensitivity.
Example 3
A dynamic dual-mode ultrasensitive immunodetection test strip and its preparation method are provided.
Example 3 is exemplified by the detection of NT-proBNP (N-terminal type B natriuretic peptide precursor).
The materials/reagents used in this example include, in parts by volume:
2 parts of MAF@AuNPs composite nano particles, 0.03 part of primary antibody protein NT-proBNP-Ab1, 0.2 part of blocking agent, 0.03 part of cosolvent and 0.05 part of working buffer.
The weight portion of the composition comprises:
2.5 parts of primary Anti-protein antibody Anti-PCT-Ab1 and 2.5 parts of secondary Anti-protein PCT-Ab2.
The preparation method comprises the following steps:
(1) Sequentially fixing the bottom plate, a water absorption pad, a chromatographic membrane, a marking pad and a sample pad on the bottom plate;
(2) Using tetra (4-carboxylbenzene) ethylene as an organic ligand and zirconium chloride as metal ion salt to synthesize MAF nano particles;
(3) Coating the nano gold particles with sodium citrate to prepare AuNPs;
(4) Mixing and incubating negatively charged MAF nano particles with 0.1% polyethylenimine-10000 for 0.5h at room temperature under stirring, centrifuging the mixed solution, removing supernatant, taking precipitate, redissolving in equal volume of deionized water to obtain surface chemically modified MAF nano particles, mixing and incubating with 2.5 parts of negatively charged AuNPs for 1h, centrifuging the mixed solution to remove supernatant, taking precipitate, redissolving in equal volume of deionized water, and self-assembling to form MAF@AuNPs composite nano particles;
(5) Mixing MAF@AuNPs composite nano particles with NT-proBNP-Ab1, placing the mixture in a shaking table for incubation for 2 hours, adding a sealing agent for further incubation for 0.5 hour, centrifuging at 8000rpm for 10 minutes, removing supernatant, taking precipitate, and redissolving the precipitate by using a redissolution to prepare the NT-proBNP-Ab1 modified MAF@AuNPs composite nano particles;
(6) Dripping 0.0025 part by volume of NT-proBNP-Ab1 modified MAF@AuNPs composite nano particles into a marking pad, and drying;
(7) Streaking the Anti-NT-proBNP-Ab1 on a chromatographic membrane, and marking the chromatographic membrane as a C line; drawing NT-proBNP-Ab2 on a chromatographic membrane, marking as T line, and drying;
(8) The sample pad was treated with borate buffer to prepare an immunodetection test strip.
The detection method comprises the following steps:
and (3) dripping a standard solution containing a target to be detected on a sample pad, dripping a corresponding working buffer on the sample pad, and respectively recording colorimetric and fluorescent signals of a T line/C line after chromatography is finished (15 min).
As shown in FIG. 3, the detection result shows that the Color mode (Color) sensitivity is 0.5ng/mL, the Fluorescence mode (Fluorescence) sensitivity is 2pg/mL (clinical threshold 300 pg/mL), and the Fluorescence mode is improved by about 250 times compared with the Color mode sensitivity.
Example 4
A dynamic dual-mode ultrasensitive immunodetection test strip and its preparation method are provided.
Example 4 exemplifies cTnI (cardiac troponin) detection.
The materials/reagents used in this example include, in parts by volume:
2 parts of MAF@AuNPs composite nano particles, 0.03 part of primary antibody protein cTnI-Ab1, 0.2 part of blocking agent, 0.3 part of cosolvent and 0.05 part of working buffer.
The weight portion of the composition comprises:
2.5 parts of primary Anti-protein antibody Anti-PCT-Ab1 and 2.5 parts of secondary Anti-protein PCT-Ab2.
The preparation method comprises the following steps:
(1) Sequentially fixing the bottom plate, a water absorption pad, a chromatographic membrane, a marking pad and a sample pad on the bottom plate;
(2) Using tetra (4-carboxylbenzene) ethylene as an organic ligand and zirconium chloride as metal ion salt to synthesize MAF nano particles;
(3) Coating the nano gold particles with sodium citrate to prepare AuNPs;
(4) Mixing and incubating negatively charged MAF nano particles with 0.1% polyethylenimine-10000 for 0.5h at room temperature under stirring, centrifuging the mixed solution, removing supernatant, taking precipitate, redissolving in equal volume of deionized water to obtain surface chemically modified MAF nano particles, mixing and incubating with 2.5 parts of negatively charged AuNPs for 1h, centrifuging the mixed solution to remove supernatant, taking precipitate, redissolving in equal volume of deionized water, and self-assembling to form MAF@AuNPs composite nano particles;
(5) Mixing MAF@AuNPs composite nano particles with cTnI-Ab1, placing the mixture in a shaking table for incubation for 2 hours, adding a sealing agent for further incubation for 0.5 hour, centrifuging at 8000rpm for 10 minutes, removing supernatant, taking precipitate, and redissolving the precipitate by using a cosolvent to prepare the cTnI-Ab1 modified MAF@AuNPs composite nano particles;
(6) Dripping 0.0025 part by volume of cTnI-Ab1 modified MAF@AuNPs composite nano particles into a marking pad, and drying;
(7) Drawing an Anti-cTnI-Ab1 line on a chromatographic membrane, and marking the chromatographic membrane as a C line; marking cTnI-Ab2 on a chromatographic membrane, marking as a T line, and drying;
(8) The sample pad was treated with borate buffer to prepare an immunodetection test strip.
The detection method comprises the following steps:
and (3) dripping a standard solution containing a target to be detected on a sample pad, dripping a corresponding working buffer on the sample pad, and respectively recording colorimetric and fluorescent signals of a T line/C line after chromatography is finished (15 min).
As shown in FIG. 4, the detection result shows that the Color mode (Color) sensitivity is 20ng/mL, the Fluorescence mode (Fluorescence) sensitivity is 10pg/mL (clinical threshold 12 pg/mL), and the Fluorescence mode is improved by about 2000 times compared with the Color mode sensitivity.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, any modification, equivalent replacement, improvement or the like of the prior art through logic analysis, reasoning or limited experiments according to the present invention will be within the scope of protection defined by the claims.
Claims (10)
1. A preparation method of a dynamic dual-mode and ultrasensitive immunodetection test strip is characterized by comprising the following steps:
(1) Sequentially fixing the bottom plate, a water absorption pad, a chromatographic membrane, a marking pad and a sample pad on the bottom plate;
(2) Using tetra (4-carboxylbenzene) ethylene as an organic ligand and zirconium chloride as metal ion salt to synthesize MAF nano particles;
(3) Coating the nano gold particles with sodium citrate to prepare AuNPs;
(4) After carrying out surface chemical modification on MAF nano particles, mixing and incubating the MAF nano particles with AuNPs for 0.5-2 h, and self-assembling to form MAF@AuNPs composite nano particles;
(5) Mixing and incubating MAF@AuNPs composite nano particles with primary antibody protein for 1-3 hours, adding a sealing agent, continuously incubating for 0.1-1 hour, centrifuging, taking precipitate, and re-dissolving by using a re-solvent to prepare Ab1 modified MAF@AuNPs composite nano particles;
(6) Dripping Ab1 modified MAF@AuNPs composite nano particles on a marking pad, and drying;
(7) Streaking an anti-protein antibody on a chromatographic membrane, and marking the chromatographic membrane as a C line; marking the secondary antibody protein on a chromatographic membrane, marking the chromatographic membrane as a T line, and drying;
(8) The sample pad was treated with borate buffer to prepare an immunodetection test strip.
2. The preparation method of claim 1, wherein in the step (5), 1 to 3 parts by volume of the maf@aunps composite nano particles are contained, 0.02 to 0.05 part by volume of the primary antibody protein is contained, 0.1 to 0.4 part by volume of the blocking agent is contained, and 0.2 to 0.5 part by volume of the complex solvent is contained.
3. The method according to claim 1, wherein in the step (7), the primary antibody is 2 to 5 parts by weight and the secondary antibody is 2 to 5 parts by weight.
4. The method of claim 1, wherein in step (4), the step of chemically modifying the surface comprises: and mixing and incubating the MAF nano particles and positively charged polymer molecules for 0.1-1 h, centrifuging, and taking the precipitate for redissolution.
5. The method according to claim 4, wherein the positively charged polymer molecule is at least one of polylysine, epsilon-polylysine, polyvinylamine, polyacrylamide, polyamide, and polyethyleneimine.
6. The method according to claim 5, wherein the molecular weight of the polyethyleneimine is 600 to 20000.
7. The method according to claim 6, wherein the mass concentration of the polyethyleneimine is 0.01 to 1%.
8. The method according to claim 1, wherein in the step (5), the blocking agent and the cosolvent are phosphate buffers containing any one of bovine serum albumin, polyethylene glycol, gelatin, and tween.
9. The immunoassay test strip prepared by the method of any one of claims 1 to 8, wherein the immunoassay test strip comprises a base plate and a water absorbing pad, a chromatographic membrane, a labeling pad and a sample pad on the base plate; the length of the immune detection test paper strip is 40-100 mm, and the width is 2-10 mm.
10. The method for detecting an immunodetection test strip of claim 9, wherein said method for detecting comprises the steps of:
and dripping the sample solution to be detected on a sample pad of the immunodetection test strip, dripping the working buffer on the sample pad, and observing a colorimetric signal of the T line/C line when the sample solution to be detected is in high concentration and observing a fluorescent signal of the T line/C line under ultraviolet light when the sample solution to be detected is in low concentration after the chromatography is finished.
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